The present invention relates to methods for isolating leukocytes and genetic materials therefrom. It also relates, in part, to a method for isolating genetic material, such as genomic DNA, from spent leukodepletion or leukoreduction filter devices in order to analyze the genetic material.
Each year in the United States about 14 million transfusions of blood or blood components take place. There are three major blood products in transfusion medicine:
1. RED BLOOD CELLS (RBC, typically about 340 ml contained in 1 unit of donor blood)xe2x80x94the remaining red cell mass after most of the plasma is removed.
2. PLATELETS (typically 300 ml/1 unit of donor blood) or platelet concentrates (PCs, typically further concentrated to about 50 ml/1 unit of donor blood)xe2x80x94one platelet concentrate (one unit of random donor platelets) is derived from one unit of donor blood.
3. FRESH FROZEN PLASMA (FFP, 225 ml/1 unit of donor blood)xe2x80x94One unit of FFP can raise coagulation factor levels by 8% and fibrinogen by 13 mg/dl in the average patient.
Despite the increasing need for transfusions and the use of transfusion products, such use involves a number of risks. About 150,000 patients each year experience adverse reactions to such products. Such adverse reactions occur regardless of the type of blood transfusion a patient receives. Ninety percent of adverse transfusion reactions are caused by donor leukocytes contained in the transfusion products.
Further problems stem from Human Leukocyte Antigen (HLA) alloimmunization, in which the recipient is sensitized to antibodies contained in the transfusion product which can react, for example, to the recipient""s leukocytes (HLA sensitization).
Where the recipient suffers from a non-hemolytic febrile transfusion reaction, the patient most frequently experiences fever, chills, and nausea due to white blood components contained in the transfusion product, to which the patients has antibodies (usually anti-HLA).
Other serious risks of the use of transfusion products include transmission and/or reactivation of cytomegalovirus (CMV), occurrence of graft-versus-host disease (GVHD), and the risk of viral transmissions. (HIV, HCV transmission are the most feared complications of transfusion.)
Certain precautions have been adopted in order to reduce the likelihood and/or severity of adverse reactions to transfusion products. Leukoreduction of blood products before transfusion into a patient is considered the most significant recent improvement in safety and purity of blood transfusion. Leukoreduction is the process of removing  greater than 99.9% of the white blood cells (WBC) from cellular blood components (red cells and platelets).
Leukodepletion (LD) (also known as leukoreduction) is a technique most commonly carried out by the filtration of whole blood or blood products to remove nucleated cells (leukocytes) from a donated sample required for transfusion. LD is a very potent measure employed by the transfusion medicine industry to avoid the risk of transferring disease from donor to patient and also as a prevention of adverse immuno-response to the donated blood.
The FDA has announced publicly that it will require that all cellular blood components transfused in the U.S be leukoreduced (leukodepleted) by the year 2002. Worldwide, ten countries, including Canada, Britain, France, Portugal, and Germany, have mandated universal leukocyte reduction, and 13 more, including Denmark, Italy, Japan, and New Zealand, are moving toward the practice.
As in any essential step of blood processing, the step of leukoreduction is subject to quality control. In order to label a component as leukocyte-reduced (leukoreduced), the American Association of Blood Bank Standards (19th ed) requires that the residual leukocyte content in the component must be  less than 5xc3x97106 WBC/unit blood. European guidelines define leukocyte reduction as residual leukocyte content of  less than 1xc3x97106 leukocytes/unit.
FDA guidelines state that quality control testing of leukocyte-reduced units should be performed on at least 1% of products (or 4/month for facilities preparing  less than 400 units/month) and that 100% of tested units are required to contain  less than 5xc3x97106 residual leukocytes/unit.
Most LD techniques employ a filter system that specifically captures leukocytes from blood, allowing the other desired blood components to pass. Specific leukocyte capture by filtration can either be carried out by affinity interaction of a leukocyte cell surface antigen such as P-selectin, CD44 or CD8, for example, or more commonly by a physical entrapment of the relatively larger leukocytes within the filter matrix of an LD device. In either case, an LD filter device, once used for the removal of leukocytes from a donated blood unit (600 ml), contains a very high concentration of leukocytes.
Potentially, assuming 100% leukodepletion, the spent LD filter device will contain between 36xc3x97108 and 6xc3x97109 cells. Each leukocyte is nucleated; i.e., it contains a nucleus that is the storage organelle for genomic DNA (gDNA), the molecular representation of an organism""s genetic makeup. Therefore, a spent LD filter device containing many leukocytes will also carry the genetic makeup of the donor.
Genotyping is the discipline of identifying an individual""s genome in relation to disease-specific alleles and/or mutations that occur as an effect of parental linkage. The rapid purification of human genomic DNA is an essential part of a genotyping process; the genomic DNA of an individual being the structural unit for the entire DNA sequence of every allele expressed.
Human genomic DNA cannot be directly sequenced. In order to carry out sequence analysis on regions of the chromosomes that may contain portions of mutation or disease specific sequences, selected portions are amplified, e.g., via polymerase chain reaction (xe2x80x9cPCRxe2x80x9d), and the amplified products are sequenced. The selected portions of the chromosomes that are amplified are dictated by the specific sequence of the primers used in the PCR amplification. The primer sets that are used in genotyping studies are commercially available and are representative for the chromosome under examination. If linkage studies identify that a disease-bearing sequence is on a particular chromosome, then many primer sets will be utilized across that chromosome in order to obtain genetic material for sequencing. The resultant PCR products may well represent the entire chromosome under examination. Due to the large length of chromosomes, many PCR reactions are carried out on the genomic DNA template from a single patient.
Human genomic DNA is currently purified by a variety of methods (Molecular Cloning, Sambrook et al. (1989)). Consequently, many commercial kit manufacturers provide products for such techniques, for example: AmpReady(trademark) (Promega, Madison, Wis.), DNeasy(trademark) (Qiagen, Valencia, Calif.), and Split Second(trademark) (Roche Molecular Biochemicals, Indianapolis, Ind.). These products rely on the use of specialized matrices or buffer systems for the rapid isolation of the genomic DNA molecule.
Recently, microporous filter-based techniques have surfaced as tools for the purification of genomic DNA as well as a whole multitude of nucleic acids. The advantage of filter-based matrices are that they can be fashioned into many formats that include tubes, spin tubes, sheets, and microwell plates. Microporous filter membranes as purification support matrices have other advantages within the art. They provide a compact, easy to manipulate system allowing for the capture of the desired molecule and the removal of unwanted components in a fluid phase at higher throughput and faster processing times than possible with column chromatography. This is due to the fast diffusion rates possible on filter membranes. Nucleic acid molecules have been captured on filter membranes, generally either through simple adsorption or through a chemical reaction between complementary reactive groups present on the filter membrane or on a filter-bound ligand resulting in the formation of a covalent bond between the ligand and the desired nucleic acid.
Porous filter membrane materials used for non-covalent nucleic acid immobilization have included materials such as nylon, nitrocellulose, hydrophobic polyvinylidinefluoride (PVDF), and glass microfiber. A number of methods and reagents have also been developed to also allow the direct coupling of nucleic acids onto solid supports, such as oligonucleotides and primers (e.g. J. M. Coull et al., Tetrahedron Lett. vol. 27, page 3991; B. A. Conolly, Nucleic Acids Res., vol. 15, page 3131, 1987; B. A. Conolly and P. Rider, Nucleic Acids Res., vol. 12, page 4485, 1985; Yang et al., P.N.A.S. vol. 95: 5462-5467). UV cross-linking of DNA (Church et al., PNAS, vol. 81, page 1991, 1984), The Generation Capture Column Kit (Gentra Systems, Minneapolis, Minn.) and RNA (Khandjian et al., Anal. Biochem, vol. 159, pages 227, 1986) to nylon membranes have also been reported.
More recently, glass microfiber, has been shown to specifically bind nucleic acids from a variety of nucleic acid containing sources very effectively (See, e.g., M. Itoh et al., Simple and Rapid Preparation of Plasmid Template by a Filtration Method using Microtiter Filter Plates, 25 Nucl. Acids Res., 1315, 1315-1316 (1997); B. Andersson et al., Method for 96-well M13 DNA Template Preparations for Large-Scale Sequencing, 20 BioTechniques 1022, 1022-1027 (1996)). Under the correct salt and buffering conditions, nucleic acids will bind to glass or silica with high specificity.
Commercially available leukodepletion filters are often made of glass fibers, polyester fibers, or a combination of the two types of fibers. One such commercially available leukodepletion filter, the r LS leukodepletion filter media (HemaSure, Inc.), for example, combines a matrix of fibers, such as glass fibers, with components, such as a highly fibrillated fibers or particles comprising a polyacrylonitrile copolymer having a specific surface area greater than 100 m2/g and an average diameter of less than 0.05 xcexcm, and, optionally, a binder, such as a polyvinyl alcohol or its derivative. This filter is capable of removing at least 99.99% of the leukocytes from a unit of blood product to provide a leukodepleted blood product. Other commercial leukodepletion filters are available from manufacturers, such as the Pall Purecell LRF High Efficiency Leukocyte Reduction Filtration System (Pall Corporation) and leukoreduction products by Baxter Healthcare Corporation (Fenwal Division)/Asahi Medical Corporation.
As the medical industry becomes more prognostic in nature, and drug companies strive to understand the genetic variations within a population set, which causes different reactions to a given drug compound, the need to study the genetic makeup of that population is essential. In order to carry out such population genetics studies (polymorphism studies), gDNA must be obtained from all the contributors of the study.
For example, New York Blood Center (xe2x80x9cNYBCxe2x80x9d) is the world""s largest blood bank currently collaborating with organizations, such as Academic Medicine Development Company (xe2x80x9cAMDeCxe2x80x9d)/North Shore University (New York, N.Y.), to carry out polymorphism studies (300,000 people per study). Currently NYBC sells units of whole blood to AMDeC for extraction of gDNA from the units. Typically, the first step of gDNA extraction from blood is to isolate cells which contain genomic DNA; this is almost always carried out by centrifugation. Centrifugation, although well established, is very time consuming and inefficient in terms of yield, and DNA shearing may also occur during centrifugation steps. The cells in whole blood which contain genomic DNA are, at least primarily, leukocytes.
Extraction of gDNA from whole blood units is both time-consuming and expensive. Moreover, the use of blood units for gDNA extraction competes with patient treatment for the already inadequate supply of whole blood at a time of increasing demand.
The present invention provides methods which utilize spent leukodepletion filter devices as a source of material for isolation and analysis of genomic DNA, including analyses for polymorphism, genotyping, and pharmacogenomic studies, as well as procedures to efficiently remove high quality intact gDNA molecules from leukodepletion filters.
According to the present invention, a method is provided for utilizing spent leukodepletion filter devices as a source for the isolation of genomic DNA for analysis.
According to the present invention, a method is provided for utilizing spent leukodepletion filter devices, comprising filters with leukocytes retained as a cellular retentate, as a source for the isolation of genomic DNA for analysis, the method comprising:
(a) providing a leukodepletion filter device having cellular retentate containing leukocytes;
(b) lysing the cellular retentate to form a cell lysate;
(c) treating the filter to remove cell lysate from the filter while retaining leukocyte nuclei with the filter;
(d) rupturing the leukocyte nuclei retained with the filter in step (c) and removing the non-nucleic acid contents of the nuclei from the filter while retaining the contents of the nuclei including nucleic acids; and
(e) eluting the nucleic acid.
In one embodiment, an LD filter device is treated with solutions which are DNase-free. The leukocytes retained with the filter (cellular retentate) are then lysed with a cell lysis agent to form a cell lysate, which is removed from the filter, while the leukocyte nuclei and contents of the nuclei, including genomic DNA, are retained by the filter. The leukocyte nuclei retained with the filter are then ruptured, preferably with a nucleus lysis agent. At least a substantial amount of nucleic acid, including genomic DNA, is retained by the filter, while the debris from the ruptured nuclei is removed. Elution of the nucleic acid is then performed, preferably using heat and a DNA liberating medium, preferably either sterile, DNase-free water or an appropriate elution buffer.
In one embodiment, the elution buffer (or water) is heated to an elevated temperature prior to addition to the filter. In another embodiment, unheated elution buffer (or water) is added to the filter, followed by heating of the filter together with the elution buffer (or water). In a preferred embodiment, the elution buffer (or water) is heated to an elevated temperature prior to addition to the filter, followed by incubation of the filter and elution buffer (or water) together at an elevated temperature that may be the same as, or different than, the elevated temperature used for the initial heating of the buffer or water.
The present invention further provides for DNA analysis kits, comprising the materials used to liberate DNA from cells retained on a leukocyte depletion filter. Preferably, the kits also comprise reagents which are used for the desired DNA analysis. For example, a preferred kit comprises a cell lysis agent, which preferably lyses cellular membranes without lysing at least a substantial portion of nuclear membranes, a nucleus lysing medium, which lyses the membrane of nuclei, a DNA liberating medium, and a set of primers for carrying out analysis of at least one target portion of the genomic DNA. The kit may include other components useful to the analysis, e.g., further purifying agents, filters or columns, reagents for carrying on DNA amplification and/or analysis, and analytical materials and/or devices for recognizing the presence of DNA sequences which are the subject of the analysis.
In a preferred embodiment, contaminating red blood cells retained with the filter are lysed and removed, such as with a red cell lysis buffer, prior to the lysis of the leukocytes retained with the filter.
It is preferred that the leukocyte nuclei be ruptured after removal of the cell lysate contaminants.
In a preferred embodiment, a method is provided for utilizing spent leukodepletion filter devices, comprising filters with leukocytes retained as a cellular retentate, as a source for the isolation of genomic DNA for analysis, the method comprising:
(a) providing a leukodepletion filter device having cellular retentate including leukocytes;
(b) lysing the cellular retentate to form a cell lysate;
(c) treating the filter to remove the cell lysate from the filter, while retaining leukocyte nuclei on the filter;
(d) rupturing the leukocyte nuclei retained with the filter in step (c) and removing the non-nucleic acid contents of the nuclei from the filter while retaining the contents of the nuclei including nucleic acids;
(e) heating an elution buffer to an elevated temperature of 40xc2x0 C. to 125xc2x0 C.;
(f) adding the elution buffer to the filter;
(g) heating the filter and elution buffer to an elevated temperature of 40xc2x0 C. to 125xc2x0 C.; and
(h) eluting the nucleic acid.
While the present method focuses on removal of the DNA of interest, which is removable by heat and elution, other methods of freeing the DNA from the filter material are also contemplated. For example, where the bonds between the molecules of interest and the filter are more chemical or immunological in nature, chemical agents which break the bonds, or antigens which supplant the bound material in the immunological bond can be used.
The present invention will now be described in further detail with reference to the accompanying Examples and to the attached Figures in which: